This invention is directed to a process for the fragment-based preparation of resin-bound cyclic peptide analogs of parathyroid hormones and analogs of parathyroid hormone-related proteins, which analogs contain at least one bridge between the side chains of two non-adjacent amino acid residues. More particularly, the invention is directed to a process for the solid-phase synthesis of such cyclic peptide analogs, and to peptide fragments useful therefor.
Cyclic peptide subunits are present in a wide variety of peptides possessing useful biological activity, including parathyroid hormone analogs and parathyroid hormone-related protein analogs, vasoactive peptide analogs, cholecystokinin analogs, tumor necrosis factor (TNF) derived peptides, calcitonin analogs, somatostatin analogs, cell adhesion modulators, growth hormone releasing factor (GRF) analogs, bradykinin antagonists, analogs of tyrosine activation motifs (TAM Mimics) and amylin agonists. Of particular interest are the cyclic peptide parathyroid hormone (hPTH) analogs and parathyroid hormone-related protein (hPTHrP) analogs.
Human parathyroid hormone (hPTH) is an 84 amino acid protein which is a major regulator of calcium homeostasis. Parathyroid hormone-related protein (hPTHrP) is a 139 to 171 amino acid protein with N-terminal homology to hPTH. The N-terminal fragments of hPTH and hPTHrP, particularly those consisting of amino acids 1-34, retain the full biological activity of the parent hormone.
hPTH(1-34) has the following amino acid sequence:
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe. (SEQ ID NO: 26)
hPTHrP(1-34) has the following amino acid sequence:
Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala. (SEQ ID NO: 27)
The biological activity of hPTH is reflected in the activation of two secondary messenger systems: G-protein coupled adenylyl cyclase (AC) and G-protein coupled and uncoupled protein kinase C (PKC) activity. The N-terminal fragments hPTH(1-34)OH and hPTH(1-31)NH2 have been demonstrated to be anabolic with respect to bone formation in humans and ovariectomized rats, respectively. This increase in bone growth has been demonstrated to be coupled with stimulation of adenylyl cyclase activity. Analogs of these N-terminal fragments have significant therapeutic potential for the treatment of physiological conditions associated with bone cell calcium regulation including hypocalcemia; osteoporosis; osteopenia; and disorders associated with osteoporosis and osteopenia such as hyperparathyroidism, hypoparathyroidism, and Cushings syndrome; glucocorticoid- and immunosuppressant-induced osteopacnia; and bone fracture and bone refracture repair.
It has also been established that deletion of up to six amino acid residues from the N-terminus of hPTH(1-34) markedly decreases the resulting analog""s ability to stimulate adenylyl cyclase while having little effect on receptor binding. Thus, analogs of hPTH(1-34) truncated by up to six amino acid residues at the N-teminus inhibit the action of PTH and are useful in the treatment of disorders characterized by an excess of PTH such as hyperparathyrodism and hyperparathyrodism-related hypercalcemia crisis, hypercalcemia of malignancy, renal failure and hypertension.
Acyclic analogs of hPTH(1-27) to (1-34) are disclosed in U.S. Pat. No. 4,086,196. Acyclic analogs of hPTH(1-34) and hPTHrP (1-34) are disclosed in U.S. Pat. No. 5,589,452. [Nle8, Nle18, Tyr34, or Phe34]hPTH(1-34) are disclosed in U.S. Pat. No. 4,656,250. [Nle8, Nle18, Tyr34]hPTH(1-34) and N-truncated derivatives thereof are disclosed in U.S. Pat. Nos. 4,771,124 and 4,423,037. Other acyclic analogs of PTH(1-34) are disclosed in U.S. Pat. Nos. 5,723,577 and 5,434,246, WO 97/02834, EPA 561 412-A1, EPA 747 817-A2, WO-94/02510, WO9603437, and WO9511988-A1. Analogs of hPTH(1-28)NH2 to hPTH(1-31)NH2 and [Leu27]hPTH(1-28)NH2 to [Leu27]hPTH(1-33)NH2 are decribed in U.S. Pat. No. 5,556,940. Acyclic antagonists of the PTH receptor including N-terminally-truncated analogs of PTH are disclosed in U.S. Pat. Nos. 5,446,130, 5,229,489, 4,771,124 and 4,423,037.
Cyclic and bicyclic analogs of hPTH and hPTHrP have been disclosed. Cyclo(Lys26-Asp30)[Leu27]hPTH(1-34)NH2 and cyclo(Lys27-Asp30)hPTH(1-34)NH2 are disclosed in U.S. Pat. No. 5,556,940. Cyclo(Lys26-Asp30)[Leu27]hPTH(1-31)NH2, cyclo(Glu22-Lys26)[Leu27]hPTH(1-31)NH2, and cyclo(Lys27-Asp30)hPTH(1-31)NH2 are decribed by Barbier, et al., J. Med. Chem. 1997, 40, 1373. Monocyclic and bicyclic derivatives of hPTH(1-34) or hPTHrP(1-34) are disclosed in patent documents WO 96/40193, DE19508672-A1, and by A. Bisello, et al., in Biochemistry 1997, 36, 3293. Cyclo(Lys13-Asp17)hPTHrP(7-34)NH2, a potent antagonist of the PTH receptor, is disclosed by M. Chorev, et al., Biochemistry 1991, 30, 5698. Also, Kanmera, et al., has described a series of amide-containing analogs of hPTHrP, Peptide Chemistry 1993: Okada, Y., ed.; Protein Research Foundation, Osaka, 1994, 321-324.xe2x80x9d WO 98/51324 discloses cyclic peptide compounds of formula I 
and pharmaceutically acceptable salts and prodrugs thereof wherein
X is selected from the group consisting of
(a) R1axe2x80x94A0xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(b) R1axe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(c) R1bxe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(d) R1axe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(e) R1axe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(f) R1axe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(g) R1axe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(h) R1axe2x80x94A8xe2x80x94A9xe2x80x94,
(i) R1axe2x80x94A9xe2x80x94, and
(j) R1axe2x80x94;
Y is selected from the group consisting of
(a) xe2x80x94R3,
(b) xe2x80x94A28xe2x80x94R3,
(c) xe2x80x94A28xe2x80x94A29xe2x80x94R3,
(d) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94R3,
(e) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94R3,
(e) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94R3,
(g) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94R3, and
(h) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94R3;
R1a is H, alkyl, aralkyl or xe2x80x94COR2;
R1b is R1a or a group of formula 
R2 is alkyl, alkenyl, alkynyl, aryl or aralkyl;
R3 is a group of formula A35xe2x80x94OR4 or A35xe2x80x94NR4R5;
R4 and R5 are independently H or lower alkyl;
R6 and R9 are independently H or alkyl;
R7 is alkyl;
R8 is H, alkyl or COR2;
R10 is H or halogen;
R11 is alkyl or aralkyl;
m is 1, 2or 3;
n is 3 or 4;
A0 is absent or a peptide of from one to six amino acid residues;
A1 is Ser, Ala, Gly or D-Pro, or an equivalent amino acid thereof;
A2 is Ala, Val or Gly, or an equivalent amino acid thereof;
A3 is Ala, Ser, Gly or D-Pro, or an equivalent amino acid thereof;
A4 is Glu, Ala or Gly, or an equivalent amino acid thereof,
A5 is Ile, His, Ala or Gly, or an equivalent amino acid thereof;
A6 is Ala, Gln, Gly or D-Pro, or an equivalent amino acid thereof;
A7 is Ala, Leu, Gly, or an equivalent amino acid thereof;
A8 is Leu, Nle, Gly or D-Pro, or an equivalent amino acid thereof;
A9 is His, Ala, D-Pro or Gly, or an equivalent amino acid thereof;
A10 is Ala, Asn, Asp, Cys, homo-Cys, Glu, Gly, Lys, Orn, Ser, Thr, D-Pro, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH [(CH2)nCO2H]COxe2x80x94;
A11 is Ala, Gly, Leu or Lys, or an equivalent amino acid thereof;
A12 is Ala or Gly, or an equivalent amino acid thereof;
A13 is Ala, Asn, Asp, Cys, homo-Cys, Glu, Gly, Lys, Orn, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A14 is Ala, Asn, Asp, Cys, homo-Cys, Glu, Gly, His, Lys, Orn, Ser, Thr, D-Pro, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A15 is Ala, Gly, Ile, D-Pro or Leu, or an equivalent amino acid thereof;
A16 is Asn, Ala, Gly, D-Pro or Gln, or an equivalent amino acid thereof;
A17 is Ala, Asn, Asp, Cys, homo-Cys, Glu, Gly, Lys, Orn, Ser, Thr, D-Pro, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A18 is Asp, Cys, homo-Cys, Glu, His, Leu, Lys, Orn, Nle, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A19 is Arg or Glu, or an equivalent amino acid thereof;
A20 is Arg or an equivalent amino acid thereof;
A21 is Arg, Asp, Cys, homo-Cys, Glu, Lys, Orn, Ser, Thr, Val, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A22 is Asp, Cys, homo-Cys, Glu, His, Lys, Orn, Phe, Ser. Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A23 is Leu, Phe or Trp, or an equivalent amino acid thereof;
A24 is Leu or an equivalent amino acid thereof;
A25 is Arg, Asp, Cys, homo-Cys, Glu, His, Lys, Orn, D-Pro, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A26 is Asp, Cys, homo-Cys, Glu, His, Lys, Orn, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A27 is Leu or Lys, or an equivalent amino acid thereof;
A28 is Ile or Leu, or an equivalent amino acid thereof;
A29 is Ala, Asp, Cys, homo-Cys, Glu, Gln, Lys, Orn, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A30 is Asp, Cys, homo-Cys, Glu, Gly, Lys, Orn, Ser, Thr, xe2x80x94NHCH[(CH2)mNH2]COxe2x80x94 or xe2x80x94NHCH[(CH2)nCO2H]COxe2x80x94;
A31 is Ile, Leu or Val, or an equivalent amino acid thereof;
A32 is His, or an equivalent amino acid thereof;
A33 is Asn or Thr, or an equivalent amino acid thereof; and
A34 is Ala or Phe, or an equivalent amino acid thereof;
A35 is absent or a peptide of from 1 to 4 amino acids; and
the side chains of at least one of the following pairs of amino acid residues, A10 and A14, A13 and A17, A14 and A18, A17 and A21, A18 and A22, A21 and A25, A25 and A29 and A26 and A30 are linked through an amide, ester, disulfide or lanthionine bond to form a bridge, and the side chain of each of the following amino acid residues, A10, A13, A14, A17, A18, A21, A22, A25, A26, A29, and A30. contributes, at most, to the formation of a single bridge; provided that when the side chains of the following pairs of amino acid acid residues, A13 and A17 or A26 and A30 are linked through an amide, disulfide or lanthionine bond to form a bridge, then the side chains of at least one of the following pairs of amino acid residues, A10 and A14, A14 and A18, A17 and A21, A18 and A22, A21 and A25 and A25 and A29 are also linked through an amide, ester, disulfide or lanthionine bond.
A preferred subset of the peptide compounds of formula I comprises the peptide compounds wherein
X is selected from the group consisting of
(a) R1axe2x80x94A0xe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(b) R1axe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(c) R1bxe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(d) R1axe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(e) R1axe2x80x94A5xe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(f) R1axe2x80x94A6xe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(g) R1axe2x80x94A7xe2x80x94A8xe2x80x94A9xe2x80x94,
(h) R1axe2x80x94A8xe2x80x94A9xe2x80x94,
(i) R1axe2x80x94A9xe2x80x94, and
(j) R1axe2x80x94;
Y is selected from the group consisting of
(a) xe2x80x94R3,
(b) xe2x80x94A28xe2x80x94R3,
(c) xe2x80x94A28xe2x80x94A29xe2x80x94R3,
(d) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94R3,
(e) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94R3,
(f) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94R3,
(g) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94R3, and
(h) xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94R3;
R1a is H, alkyl, aralkyl or xe2x80x94COR2;
R1b is R1a or a group of formula 
R2 is alkyl, alkenyl, alkynyl, aryl or aralkyl;
R3 is a group of formula A35xe2x80x94OR4 or A35xe2x80x94NR4R5;
R4 and R5 are independently H or lower alkyl;
R6 and R9 are independently H or alkyl;
R7 is alkyl;
R8 is H, alkyl or COR2;
R10 is H or halogen;
R11 is alkyl or aralkyl;
A0 is absent or a peptide of from one to six amino acid residues;
A1 is Ser, Ala, Gly or D-Pro, or an equivalent amino acid thereof;
A2 is Ala, Val or Gly, or an equivalent amino acid thereof;
A3 is Ala, Ser, Gly or D-Pro, or an equivalent amino acid thereof;
A4 is Glu, Ala or Gly, or an equivalent amino acid thereof;
A5 is Ile, His, Ala or Gly, or an equivalent amino acid thereof;
A6 is Ala, Gln, Gly or D-Pro, or an equivalent amino acid thereof,
A7 is Ala, Leu or Gly, or an equivalent amino acid thereof;
A8 is Leu, Nle, Gly or D-Pro, or an equivalent amino acid thereof;
A9 is His, Ala, Gly or D-Pro, or an equivalent amino acid thereof,
A10 is Ala, Asn, Gly Lys, Asp or D-Pro, or an equivalent amino acid thereof;
A11 is Ala, Gly, Leu or Lys, or an equivalent amino acid thereof;
A12 is Ala or Gly, or an equivalent amino acid thereof;
A13 is Ala, Gly or Lys, or an equivalent amino acid thereof;
A14 is Ala, Gly, His, Ser, Asp, Lys or D-Pro, or an equivalent amino acid thereof;
A15 is Ala, Gly, Ile, D-Pro or Leu, or an equivalent amino acid thereof;
A16 is Asn, Ala, Gly, D-Pro or Gln, or an equivalent amino acid thereof;
A17 is Ala, Asp, Gly, Ser, Lys or D-Pro, or an equivalent amino acid thereof;
A18 is Lys, or an equivalent amino acid thereof;
A19 is Arg or Glu, or an equivalent amino acid thereof;
A20 is Arg, or an equivalent amino acid thereof;
A21 is Arg, Lys, Asp or Val, or an equivalent amino acid thereof;
A22 is Asp, Lys, Orn or Glu, or an equivalent amino acid thereof;
A23 is Leu, Phe or Trp, or an equivalent amino acid thereof;
A24 is Leu, or an equivalent amino acid thereof;
A25 is Arg, His, Asp, Lys or Glu, or an equivalent amino acid thereof;
A26 is Lys or His, or an equivalent amino acid thereof;
A27 is Leu or Lys, or an equivalent amino acid thereof;
A28 is Ile or Leu, or an equivalent amino acid thereof;
A29 is Ala, Asp, Glu or Gln, or an equivalent amino acid thereof;
A30 is Asp, Lys or Glu, or an equivalent amino acid thereof;
A31 is Ile, Leu or Val, or an equivalent amino acid thereof;
A32 is His, or an equivalent amino acid thereof;
A33 is Asn or Thr, or an equivalent amino acid thereof; and
A34 is Ala or Phe, or an equivalent amino acid thereof; and
A35 is absent or a peptide of from 1 to 4 amino acids.
The foregoing peptide compounds are disclosed in WO 98/51324 to possess useful properties, more particularly pharmaceutical properties. They are especially useful for treating disease states capable of being modulated by compounds which bind to parathyroid hormone receptors either with or without comcommitant stimulation of adenylyl cyclase activity. The present invention is directed to an improved method for synthesizing these peptide compounds.
The solid phase synthesis of cyclic peptides typically involves the sequential addition of amino acids to a peptide synthesis resin to obtain a resin-bound peptide possessing all or a portion of the amino acid sequence of the desired cyclic peptide. The amino acid side chain residues to be cyclized are then deprotected and cyclization is effected. If cyclization is effected prior to completion of the entire amino acid sequence, the remaining amino acids are added and the completed peptide is then cleaved from the resin and purified.
However, the linear approach to the preparation of peptides, and to cyclic peptides in general, is frequently inefficient and may therefore not be cost effective for the preparation of large quantities of peptide. In cases where a peptide is assembled in a linear fashion, the ease of purification of the peptide decreases as the number of amino acid residues in the peptide increases. Furthermore, for cyclic peptides, preparing side-chain bridge(s) near the end of the synthesis imparts the problems associated with preparing the side-chain bridge, including low yield, side-reactions and removal of impurities, to the entire peptide. Consequently, the incorporation of a cyclic unit in a lengthy peptide increases the difficulties inherent in linear peptide syntheses. A commercially feasible synthesis of cyclic hPTH analogues such as those disclosed in WO 98/51324 and set forth above requires a synthetic approach which overcomes both the complicating factors of of length and cyclic components and which would enable preparation of commercially useful quantities in the context of drug manufacturing. The present invention is directed to a more efficient preparation of cyclic hPTH and hPTHrP analogues via a process using bridged and non-bridged fragments.
This invention is directed to a method of preparing cyclic hPTH and hTPHrP analogs of formula 
wherein
J, L, and M are linear peptide fragments
K1 is absent or a cyclic peptide fragment, and
K2 is a cyclic peptide fragment;
this method comprising the steps of:
(1) preparing 
by sequential attachment of suitably protected amino acid residues to a resin to provide: 
where 
is a suitable peptide synthesis resin and M is a polypeptide fragment
(2) preparing separately by conventional peptide synthesis N-terminal protected cyclic polypeptide fragment of formula IV 
wherein P is a suitable amine protecting group,
(3) Coupling III with IV to provide a peptide of formula V 
(4) when cyclic peptide K1 is absent, then (a) polypeptide fragments J and L are prepared as a single polypeptide of formula VI 
and polypeptide VI is coupled to peptide V to provide a peptide of formula VII 
or, optionally, (b) the protected individual amino acid elements of polypeptides J and L are added sequentially to the peptide fragment of formula V,
or, optionally, (c) either or both J and L are prepared separately as polypeptide fragments and coupled to the growing peptide starting with the fragment of formula V,
(5) when cyclic peptide K1 is present, then (a) a polypeptide fragment of formula VIII 
wherein
P is a suitable amine protecting group,
is prepared separately by conventional peptide synthesis procedures,
(b) a peptide fragment of formula IX 
is prepared and coupled with peptide fragment V to provide a peptide fragment of formula X 
and (c) the cyclic peptide fragment VIII is coupled with the peptide fragment of formula X to provide a peptide fragment of formula XI 
(d) peptide fragment of formula XII is prepared 
and fragment XII is coupled with fragment XI, and
(6) cleaving the resin and deprotecting.
In the process described herein, cyclic peptide fragments are prepared separately for coupling with a resin or resin-bound peptide. The separate preparation of the cyclic peptide fragment allows for the convergent synthesis of resin bound cyclic peptides, resulting in increased yield and throughput of the resulting cyclic peptide. Difficulties associated with preparing the bridged fragment are confined to a smaller peptide subunit, which can be purified prior to coupling with the solid phase synthesis resin. The practice of this technique in the context of certain preferred hPTH and hPTHrP analogs requires the synthesis of new peptide fragments and new sequences of events.
As used above and throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
Definitions of Terms
xe2x80x9cAlkylxe2x80x9d means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain. Branched means that one or more lower alkyl groups are attached to a linear alkyl chain. xe2x80x9cLower alkylxe2x80x9d means about 1 to 4 carbon atoms in the chain which may be straight or branched. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, and the like.
xe2x80x9cAlkenylxe2x80x9d means aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched having about 2 to about 20 carbon atoms in the chain. xe2x80x9cLower alkenylxe2x80x9d means about 2 to 4 carbon atoms in the chain which may be straight or branched. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl and decenyl.
xe2x80x9cAlkynylxe2x80x9d means aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched having about 2 to about 20 carbon atoms in the chain. xe2x80x9cLower alkynylxe2x80x9d means about 2 to 4 carbon atoms in the chain which may be straight or branched. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 3-methylbut-2-ynyl, n-pentynyl, heptynyl, octynyl and decynyl.
xe2x80x9cAlkylenexe2x80x9d means a divalent group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, for example methylene, 1,2-ethylene, 1,1-ethylene, 1,3-propylene, 2,2-dimethylpropylene, and the like.
xe2x80x9cPhenylalkylxe2x80x9d means a phenyl group attached to the parent molecular moiety through an alkylene group. The alkylene group is preferably of about 1 to about 7 carbon atoms. Representative phenylalkyl groups include benzyl, 2-phenylethyl, 2-propylphenyl, and the like.
xe2x80x9cAmine protecting groupxe2x80x9d means an easily removable group which is known in the art to protect an amino group against undesirable reaction during synthetic procedures and to be selectively removable. The use of N-protecting groups is well known in the art for protecting groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, cf. for example, T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, New York (1991), incorporated herein by reference. Preferred N-protecting groups are acyl, including formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl and the like, and acyloxy including methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethxoycarbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC, or Boc), 1,1-dimethylpropynyloxycarbonyl, benzyloxycarbonyl (CBZ), p-nitrophenylsulfinyl, p-nitrobenzyloxycarbony, 2,4-dichlorobenzyloxycarbonyl, allyoxycarbonyl (Alloc), and the like.
xe2x80x9cAmino acidxe2x80x9d means an amino acid selected from the group consisting of natural and unnatural amino acids as defined herein. The amino acids may be neutral, positive or negative depending on the substituents in the side chain. xe2x80x9cNeutral amino acidxe2x80x9d means an amino acid containing uncharged side chain substituents. Exemplary neutral amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine and cysteine. xe2x80x9cPositive amino acidxe2x80x9d means an amino acid in which the side chain substituents are positively charged at physiological pH. Exemplary positive amino acids include lysine, arginine and histidine. xe2x80x9cNegative amino acidxe2x80x9d means an amino acid in which the side chain substituents bear a net negative charge at physiological pH. Exemplary negative amino acids include aspartic acid and glutamic acid. Preferred amino acids are xcex1-amino acids. The most preferred amino acids are xcex1-amino acids having L stereochemistry at the xcex1-carbon.
xe2x80x9cAmino acid residuexe2x80x9d means the individual amino acid units incorporated into a peptide or peptide fragment.
xe2x80x9cNatural amino acidxe2x80x9d means an xcex1-amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid and glutamic acid.
xe2x80x9cUnnatural amino acidxe2x80x9d means an amino acid for which there is no nucleic acid codon. Examples of unnatural amino acids include, for example, the D-isomers of the natural a-amino acids as indicated above; Aib (aminobutyric acid), bAib (3-aminoisobutyric acid), Nva (norvaline), xcex2-Ala, Aad (2-aminoadipic acid), bAad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba (xcex3-aminobutyric acid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutryic acid), xcex1-aminopimelic acid, TMSA (trimethylsilyl-Ala), aIle (allo-isoleucine), Nle (norleucine), tert-Leu, Cit (citrulline), Orn, Dpm (2,2xe2x80x2-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), xcex1- or xcex2-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), and the like; cyclic amino acids; Nxcex1-alkylated amino acids such as MeGly (Nxcex1-methylglycine), EtGly (Nxcex1-ethylglycine) and EtAsn (Nxcex1-ethylasparagine); and amino acids in which the xcex1-carbon bears two side-chain substituents.
xe2x80x9cEquivalent amino acidxe2x80x9d means an amino acid which may be substituted for another amino acid in the peptides according to the invention without any appreciable loss of function. In making such changes, substitutions of like amino acids is made on the basis of relative similarity of side chain substituents, for example regarding size, charge, hydrophilicity, hydropathicity and hydrophobicity as described herein.
As detailed in U.S. Pat. No. 4,554,101, incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (xe2x88x920.5); Thr (xe2x88x920.4); Ala (xe2x88x920.5); His (xe2x88x920.5); Cys (xe2x88x921.0); Met (xe2x88x921.3); Val (xe2x88x921.5); Leu (xe2x88x921.8); Ile (xe2x88x921.8); Tyr (xe2x88x922.3); Phe (xe2x88x922.5); and Trp (xe2x88x923.4). It is understood that an amino acid residue can be substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0) and still obtain a biologically equivalent polypeptide.
In a similar manner, substitutions can be made on the basis of similarity in hydropathic index. Each amino acid residue has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those hydropathic index values are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (xe2x88x920.4); Thr (xe2x88x920.7); Ser (xe2x88x920.8); Trp (xe2x88x920.9); Tyr (xe2x88x921.3); Pro (xe2x88x921.6); His (xe2x88x923.2); Glu (xe2x88x923.5); Gln (xe2x88x923.5); Asp (xe2x88x923.5); Asn (xe2x88x923.5); Lys (xe2x88x923.9); and Arg (xe2x88x924.5). In making a substitution based on the hydropathic index, a value of within plus or minus 2.0 is preferred.
xe2x80x9cPeptidexe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d mean a polymer in which the monomers are amino acid residues joined together through amide bonds. Preferred peptides of the present invention are those comprising xcex1-amino acids. The most preferred peptides of the present invention comprise xcex1-amino acids having L stereochemistry at the xcex1-carbon.
xe2x80x9cCyclic peptidexe2x80x9d means a peptide containing one or more cyclic peptide fragments as defined herein.
xe2x80x9cPeptide fragmentxe2x80x9d means a peptide subunit of the parent peptide. xe2x80x9cPeptide fragmentxe2x80x9d may imply a linear, branched, or cyclic subunit of the target peptide.
xe2x80x9cLinear Peptidexe2x80x9d means a peptide or polypeptide in which the amino acids are linked to one another via an amid bond between the N-terminal of one and the C-terminal or another.
xe2x80x9cBranched peptidexe2x80x9d means a peptide or polypeptide within which one or several constituent individual amino acids bearing carboxylic acid or amine side chains are attached to another peptide substituent via these side chains.
xe2x80x9cCyclic peptide fragmentxe2x80x9d means a peptide fragment as defined herein in which a substituent on one amino acid residue is linked to a substituent on another amino acid residue in the peptide fragment. The linking is preferably between the side chains of two amino acid residues in the peptide fragment, preferably through an ester, amide, disulfide or lanthionine bond. The bonding between the two amino acid side chains is designated herein as 
The ester, amide, disulfide or lanthionine bond which links two amino acid residues of the cyclic peptide is formed between the side-chain functionalities. Thus, an amide is bond is formed between the side-chain carboxyl group of an acidic amino acid residue and the side-chain amino group of a basic amino acid residue; ester bonds are formed between the side-chain carboxyl group of an acidic amino acid residue and the side-chain hydroxyl group of a hydroxyl-containing amino acid residue; disulfides are formed from amino acid residues containing side chain sulfhydryl groups; and lanthionine bridges are formed by desulfurization of the corresponding disulfide.
The number of atoms in the bridge resulting from the amide, ester, disulfide or lanthionine bond formed as described above will vary depending on the length of the side chain and the type of bond (ie, amide, ester, disulfide or lanthionine). The bridge preferably comprises from 2 to 12 atoms, more preferably from 6 to 10 atoms. The most preferred number of atoms contained in the bridge is 7, this bridge preferably comprising an amide bond between the side-chain functionalities of a Lys and an Asp residue.
xe2x80x9cResinxe2x80x9d means a solid support modified with a reactive group such that the solid support is amenable to coupling with the carboxy or N-terminus of an amino acid, peptide, or cyclic peptide fragment as defined herein. Representative resins include Merrifield resin (chloromethylated polystyrene), hydroxymethyl resin, 2-chlorotrityl chloride resin, trityl chloride resin, Rink acid resin (4-benzyloxy-2xe2x80x2,4xe2x80x2-dimethoxybenzhydrol resin), trityl alcohol resin, PAM resin (4-hydroxymethyl-phenylacetamidomethyl resin), Wang resin (p-benzyloxybenzyl alcohol resin), MBHA resin (p-methylbenzhydrylamine resin), BHA resin (benzyhydrylamine resin), Rink amide resin (4-(2xe2x80x2,4xe2x80x2-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin) and PAL resin (5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid-MBHA resin). Preferred resins are chlorotrityl resin, Rink acid resin and Rink amide resin.
xe2x80x9cSolid supportxe2x80x9d means a substrate which is inert to the reagents and reaction conditions described herein, as well as being substantially insoluble in the media used. Representative solid supports include inorganic substrates such as kieselguhr, silica gel, and controlled pore glass; organic polymers including polystyrene, including 1-2% copolystyrene divinyl benzene (gel form) and 20-40% copolystyrene divinyl benzene (macro porous form), polypropylene, polyethylene glycol, polyacrylamide, cellulose, and the like; and composite inorganic/polymeric compositions such as polyacrylamide supported within a matrix of kieselguhr particles. See J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd. Ed., Pierce Chemical Co. (Chicago, Ill., 1984).
In addition,xe2x80x9csolid supportxe2x80x9d includes a solid support as described above which is affixed to a second inert support such as the pins described in Technical Manual, Multipin(trademark) SPOC, Chiron Technologies (1995) and references therein which comprise a detachable polyethylene- or polypropylene-based head grafted with an amino functionalized methacrylate copolymer and an inert stem.
In addition, xe2x80x9csolid supportxe2x80x9d includes polymeric supports such as the polyethylene glycol supports described by Janda et al., Proc. Natl. Acad. Sci. USA, 92, 6419-6423 (1995) and S. Brenner, WO 95/16918, which are soluble in many solvents but can be precipitated by the addition of a precipitating solvent.
The names of natural and unnatural amino acids and residues thereof used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in xe2x80x9cNomenclature of xcex1-Amino Acids (Recommendations, 1974)xe2x80x9d Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and residues thereof employed in this specification and appended claims differ from those noted, differing names and abbreviations will be made clear.
A representative peptide prepared according to the process of this invention is denoted, for example, as cyclo(Lys18-Asp22)[Ala1, Nle8, Lys18, Asp22,Leu27]hPTH(1-31)NH2 with the linked amino acid residues in the parenthesis following xe2x80x9ccycloxe2x80x9d and with substituted amino acids from the natural sequence placed in brackets. hPTH stands for human parathyroid hormone, and hPTHrP for human parathyroidhormone-related protein. The numbers in the second parenthesis refer to the number of amino acid residues in the peptide, beginning at the N-terminus (ie, the first 31 amino acids of hPTH).
Preferred Embodiments
As shown in Scheme 1, the preparation of a resin-bound cyclic peptide involves coupling cyclic and noncyclic peptide fragments to a resin, resin bound amino acid, or resin bound peptide. 
is a resin suitable for peptide synthesis.
T is an H2N, and amino acid, or a cyclic or acyclic peptide.
E and Q are each or independently an N terminal protected amino acid, or an N-terminal protected cyclic or acyclic peptide fragment.
The coupling is preferably accomplished between the carboxy terminus of the peptide fragment and the resin, resin-bound amino acid or resin bound peptide. When the peptide fragment is coupled to a resin-bound amino acid or peptide, the coupling is preferably through an amide bond between the carboxy terminus of the cyclic peptide fragment and the N-terminus of the resin-bound amino acid or peptide.
In order for the coupling reaction to proceed, the carboxyl group of the peptide fragment must be activated. Many methods of activation may be used in the practice of the invention and include, for example, preformed symmetrical anhydrides (PSA), preformed mixed anhydride (PMA), acid chlorides, active esters, and in situ activation of the carboxylic acid, as set forth in Fields and Noble, 1990, xe2x80x9cSolid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acidsxe2x80x9d, Int. J. Pept. Protein Res. 35:161-214.
Representative activating agents include isopropyl chloroformate, diisopropylcarbodiimide (DIC), DIC admixed with 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP-Cl), benzotriazole-1-yloxy-tris((dimethylamino)phosphonium)hexafluorophosphate (BOP), benzotriazole-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBROP), O-(7-azabenzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazole-1-yl)-1.1.3.3-tetramethyluronium tetrafluoroborate (TBTU), 2-(1H-benzotriazole-1-yl)-1.1.3.3-tetramethyluronium hexafluorophosphate (HBTU), 2-[2-oxo-1-(2H)-pyridyl]-1,1,3,3-bispentamethyleneuronoium tetrafluoroborate (TOPPipU), N,Nxe2x80x2-dicyclohexylcarbodiimide (DCC), DCC admixed with HOBT, and the like. Suitable solvents for the coupling reaction include dichloromethane, DMF, DMSO, toluene, THF, and the like. Coupling times range from about 1 to about 48 hours, depending upon the resin and carboxylic acid derivative to be coupled, activating agent, solvent and temperature. The coupling is accomplished at from about xe2x88x9210xc2x0 C. to about 50xc2x0 C., preferably at about ambient temperature.
In order to prevent interference with the above-described coupling reaction, the N-terminus of the cyclic peptide fraction is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of amide bond formation, while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (FMOC or Fmoc), t-butyloxycarbonyl (BOC or Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (xcex1,xcex1)dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is preferred.
Likewise, it may be necessary to protect any reactive side-chain functional groups in the cyclic peptide fragment. Particularly preferred side chain protecting groups are, for side chain amino groups as in lysine and arginine: 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc, PMC or pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc, Alloc (allyloxycarbonyl) and adamantyloxycarbonyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine: t-butyl, benzyl and tetrahydropyranyl; for histidine: trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan: formyl and Boc; for asparagine and glutamine: Trt (trityl); for aspartic acid and glutamic acid: O-t-Bu and OAllyl and Obenzyl.
Coupling of the fragments to the resin or resin bound peptide is preferably accomplished at about ambient temperature using about 2 molar equivalents (relative to resin) of the Fmoc-protected cyclic peptide fragment, activated with about an equimolar portion of benzotriazolyloxy-tris[pyrrolidino]-phosphonium hexafluoroacetate (PyBOP) and hydroxybenzotriazole hydrate in the presence of about 4 equivalents of diisopropylethyl amine in dimethylformamide over about 16-48 hours.
The completeness of coupling should be assessed. Those skilled in the art would be familiar with the well known monitoring tests such as ninhydrin (the Kaiser test), picric acid, 2,4,6-trinitro-benzenesulfonic (TNBS), fluorescamine, and chloranil, which are based on reagent reaction with free amino groups to produce a chromophoric compound. If imino acids (e.g., Pro and Hyp) are used, isatin monitoring is a preferred method. Fields and Noble, supra. Quantification of reaction completeness may be monitored during the course of the reaction, e.g., as described by Salisbury et al. (International Patent Publication No. WO91/03485).
With Fmoc synthesis, the Kaiser and TNBS tests are preferred. In the Kaiser test, a sample of resin peptide can be tested with ninhydrin reagent obtained from Pierce Chemical in the method set forth by Sarin et al. (1981, Anal. Biochem. 117:147-157.). Similarly, a sample of resin peptide L can be tested using trinitrobenzenesulfonic acid (TNBS), available from Fluka, as set forth by Takajashi (1984, Chem Lett. 1: 127)
If the coupling reaction is incomplete as determined by one or the other of these tests, the reaction can be forced to completion by several methods familiar to those in the art, including (a) a repeat coupling using a one to five fold excess of protected amino acid, (b) a repeat coupling using different or additional solvents (e.g., trifluoroethane), or (c) the addition of chaotropic salts, e.g., NaCIO4 or LiBr (Klis and Stewart, 1990, xe2x80x9cPeptides: Chemistry, Structure and Biology,xe2x80x9d Rivier and Marshall, eds., ESCOM Publ., p. 904-906).
A preferred resin-bound cyclic peptide fragment has formula II 
wherein 
is a resin;
P is an amine protecting group;
A is absent or a group of formula 
B is a group of formula xe2x80x94A23xe2x80x94A24xe2x80x94A25xe2x80x94;
C is a group of formula xe2x80x94A26xe2x80x94A27xe2x80x94A28xe2x80x94A29xe2x80x94A30xe2x80x94;
and
D is xe2x80x94A31xe2x80x94, xe2x80x94A31xe2x80x94A32xe2x80x94, xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94 or xe2x80x94A31xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94;
wherein 
is a lactam, ester, disulfide or lanthionine bridge; A13, A17, A18 and A22 are amino acid residues; A14 is His(Trt) or Ser(tBu); A15 and A28 are independently Ile or Leu; Ser(tBu) or Leu; A16 is Asn(Trt) or Gln(Trt); A19 is Arg(Pmc) or Glu(OtBu); A20 is Arg(Pmc); A21, is Arg(Pmc) or Val; A23 is Phe or Trp(Boc); A24 is Leu; A25 is Arg(Pmc) or His(Trt); A26 and A30 are amino acid residues wherein the side chains of A26 and A30 are optionally linked through an amide, disulfide or lanthionine bond; A27 is Leu or Lys(Boc); A29 is Ala or Gln(Trt); A31 is Ile or Val; A32 is His(Trt); A33 is Asn(Trt) or Thr(tBu); and A34 is Ala or Phe.
Another preferred resin-bound cyclic peptide fragment has formula II wherein 
is a lactam bridge and the side chains of A26 and A30 are optionally linked through an amide bond.
Another preferred resin-bound cyclic peptide has formula II wherein 
is a lactam bridge; A13 and A18 are Lys; A17 and A22 are Asp; and A26 is Lys(Boc); and A30 is Asp(OtBu); or the side chains of A26 and A30 are optionally linked through an amide bond.
Another preferred resin-bound cyclic peptide fragment has formula III 
wherein 
is a lactam bridge;
A18 is Lys; A19 is Glu(OtBu); A20 is Arg(Pmc); A21 is Val; A22 is Asp, A23 is Trp(Boc); A24 is Leu; A25 is Arg(Pmc); A26 is Lys(Boc); A27 is Leu; A28 is Leu; A29 is Gln(Trt); A30 is Asp(OtBu); and A31 is Val.
The resin-bound peptide prepared as described above may be further elaborated, for example by coupling with one or more additional cyclic peptide fragments, by coupling with one or more peptide fragments, by sequential addition of individual amino acids, or by any combination of the foregoing.
The complete cyclic peptide is then cleaved from the resin and purified. Protecting groups may be removed prior or subsequent to, or simultaneously with, cleavage from the resin. The fully deprotected peptide may purified, alone or in combination, by acid-base extraction, recrystallization, lyophilization, by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (for example, AMBERLITE(copyright) XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on SEPHADEX(copyright) G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
Preparation of the Peptide Fragment
The fragment is prepared by synthesizing the peptide backbone, which entails assembling the amino acids comprising the fragment in their proper order. The peptide backbone of the fragment may be synthesized by any of the techniques that are known to those skilled in the art. For solid phase peptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W.H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. 1, Acacemic Press (New York), 1965.
As would be known to those of ordinary skill in the art, the process of peptide synthesis on solid supports generally involves building a peptide from the carboxyl or C-terminal end in which the C-terminal amino acid with its ax-amino group protected is attached to a solid phase polymer. The N-protecting group is then cleaved off, and the next amino acid, also N-protected, is coupled by a peptide bond to the xcex1-amino group of the amino acid attached to the solid support as described above. The cycle of deprotection of the prior amino acid and coupling the additional amino acid is repeated until the peptide is completed. Any reactive side chains of the amino acids are protected by chemical groups that can withstand the coupling and Nxcex1-deprotection procedure but can be removed at the end of the synthesis. When the peptide fragment is prepared using solid phase methods, the fragment is obtained for use in couplings to other fragments by cleavage from the resin.
A cyclic fragment is prepared in similar fashion. However, backbone assembly is followed by (1)selective deprotection of the side-chain functionalities to be cyclized, (2)cyclization, and (3)optional removal of any remaining protecting groups.
The use of Fmoc amino acids is but one strategy of peptide synthesis. A Boc (t-butyloxycarbonyl-protected amino group) strategy may also be used to prepare a peptide bound to the solid support (e.g., Geysen et al., 1987, J. Immunol. Methods 102:259-274.)
Amino acids used for peptide synthesis may be standard Boc (Nxcex1-amino protected Nxcex1-t-butyloxycarbonyl) amino acids described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile Nxe2x96xa1-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc Nxe2x96xa1-amino protected amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to those who practice this art. In addition, the method of the invention can be used with other Nxe2x96xa1-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or using automated synthesizers, such as sold by ABS.
Although C-terminal to N-terminal peptide synthesis is the standard, those of ordinary skill in the art will recognize that the peptide fragment is also amenable to synthesis from N-terminus to C-terminus.
Preparation of the Lactam-Bridge
Lactam-bridged cyclic peptide fragments are prepared by formation of an amide bond between the side-chain carboxyl group of an acidic amino acid residue and the side-chain amino group of a basic amino acid residue in the presence of an activating agent as described above. Preferred acidic amino acid residues include Asp, Glu, xe2x80x94NHCH[(CH2)3CO2H]COxe2x80x94 and xe2x80x94NHCH[(CH2)4CO2H]COxe2x80x94, Asp being most preferred. Preferred basic amino acid residues include His, Lys, Orn, xe2x80x94NHCH(CH2NH2)COxe2x80x94 and xe2x80x94NHCH[(CH2)2NH2]COxe2x80x94, Lys being most preferred.
In instances wherein the peptide precursor to the cyclic peptide fragment contains more than one acidic or basic amino acid residue, protecting groups for the additional acidic or basic amino acids in the cyclic peptide fragment are selected so that the amino acids to be cyclized may be selectively deprotected. Preferably, the desired acidic and basic amino acid residues are deprotected simultaneously. Furthermore, in addition to being stable to the reagents used to deprotect the selected basic and acidic amino acid residues, the protecting groups on the remaining amino acid residues are selected to be stable to the cyclization conditions employed.
The term xe2x80x9corthogonalityxe2x80x9d when used in reference to side chain protecting groups refers to a situation as described herein in which there are two or more classes of protecting groups on a molecule, each class most optimally removed under specific conditions, while remaining stable to conditions used to remove protecting groups in other classes. Thus one can remove all protecting groups of one class, while leaving all others intact.
Preferred protecting groups having the desired orthogonality are: for the acidic amino acid residue to be cyclized: allyl; for the basic amino acid residue to by cyclized: allyloxycarbonyl (alloc); for any additional acidic amino acid residues: tert-butyl (tBu); and for any additional basic amino acid residues: tert-butyloxycarbonyl (Boc).
Following synthesis of the peptide backbone of the cyclic peptide fragment using solid phase or solution phase techniques, the allyl and allyloxycarbonyl protecting groups are removed simultaneously by treatment with palladium, preferably tetrakis(triphenylphosphine) palladium(0) in the presence of N-methylaniline. Formation of the lactam bridge is then accomplished as described herein for amide bond formation.
Preferred cyclic peptide fragments for use in preparing the resin-bound cyclic peptides of this invention have formula XV, XVI or XVII 
wherein 
is a lactam, disulfide or lanthionine bridge;
P is an amine protecting group;
A13, A17, A18, A22, A26 and A30 are amino acid residues; A14 is His(Trt) or Ser(tBu); A15 is Ser(tBu) or Leu; A16 is Asn(Trt) or Gln(Trt); A19 is Arg(Pmc) or Glu(OtBu); A20 is Arg(Pmc); A21 is Arg(Pmc) or Val; A27 is Leu or Lys(Boc); A28 is Ile or Leu; and A29 is Ala or Gln(Trt).
More preferred cyclic peptide fragments have formula IV, V or VI wherein 
is a lactam bridge;
P is Fmoc;
A13, A18 and A26 are Lys; A17, A22 and A30 are Asp; A14 is His(Trt) or Ser(tBu); A15 is Ser(tBu) or Leu; A16 is Asn(Trt) or Gln(Trt); A19 is Arg(Pmc) or Glu(OtBu); A20 is Arg(Pmc); A21 is Arg(Pmc) or Val; A27 is Leu or Lys(Boc); A28 is Ile or Leu; and A29 is Ala or Gln(Trt).
A still more preferred cyclic peptide fragment for use in preparing the resin-bound cyclic peptides of this invention is Fmoc-cyclo(Lys-Asp)Lys-Glu(OtBu)-Arg(Pmc)-Val-Asp-OH.
Cyclic peptide parathyroid hormone analogs and parathyroid hormone-related protein analogs suitable for preparation using the process of this invention are disclosed by:
1. Condon et al., U.S. Ser. No. 60/046,472 (filed May 14, 1997);
2. Willick et al., U.S. Pat. No.: 5,556,940 (Sep. 17, 1997);
3. Chorev et al., U.S. Pat. No. 5,717,062 (Feb. 10, 1998);
4. Vickery et al., WO 96/40775 (published Dec. 19, 1996).
A preferred cyclic peptide suitable for preparation using the process of this invention has formula I 
wherein
X is selected from the group consisting of (a) R1axe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94 (b) R1bxe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (c) R1bxe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (d) R1bxe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (e)R1bxe2x80x94A5xe2x80x94A6xe2x80x94, (f) R1bxe2x80x94A6xe2x80x94 and (g) R1bxe2x80x94 wherein R1a is selected from the group consting of (1) H, (2) a peptide comprising from 1 to 6 amino acids, (3) alkyl, (4) phenylalkyl, (5) xe2x80x94COR2 wherein R2 is selected from alkyl, alkenyl, alkynyl, phenyl, naphthyl and phenylalkyl, R1b is selected from the group consting of (1) H, (2) alkyl, (3) phenylalkyl and (4) xe2x80x94COR2 wherein R2 is selected from alkyl, alkenyl, alkynyl, phenyl, naphthyl and phenylalkyl, A1 is selected from Ser and Ala or an equivalent amino acid residue thereof, A2, A3, A4 and A6 are amino acid residues and A5 is selected from Ile and His or an equivalent amino acid residue thereof;
A7, A9, A11 and A12 are amino acid residues;
A8 is Leu or Nle or an equivalent amino acid residue thereof;
A10 is Asp or Asn or an equivalent amino acid residue thereof;
A13 and A17 are amino acid residues wherein the side chains of A13 and A17 are linked through an amide bond;
A14 is His or Ser or an equivalent amino acid residue thereof;
A15 and A28 are independently Ile or Leu or an equivalent amino acid residue thereof.
A16 is Asn or Gln or an equivalent amino acid residue thereof;
A18 and A22 are amino acid residues wherein the side chains of A18 and A22 are linked by an amide bond;
A19 is Arg or Glu or an equivalent amino acid residue thereof;
A20 is Arg or an equivalent amino acid residue thereof;
A21 is Arg or Val or an equivalent amino acid residue thereof;
A23 is Phe or Trp or an equivalent amino acid residue thereof;
A24 is Leu or an equivalent amino acid residue thereof;
A25 is Arg or His or an equivalent amino acid residue thereof;
A26 and A30 are amino acid residues wherein the side chains of A26 and A30 are optionally linked through an amide bond;
A27 is Leu or Lys or an equivalent amino acid residue thereof;
A29 is Ala or Gln or an equivalent amino acid residue thereof;
A31 is Ile or Val or an equivalent amino acid residue thereof;
Y is selected from the group consisting of (a) R3, (b) A32xe2x80x94R3, (c) xe2x80x94A32xe2x80x94A33xe2x80x94R3 and (d)xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94R3 wherein R3 is xe2x80x94OH or xe2x80x94NR4R5 wherein R4 and R5 are independently from hydrogen and lower alkyl, A32 is His or an equivalent amino acid residue thereof, A33 is Asn or Thr or an equivalent amino acid residue thereof and A34 is Ala or Phe or an equivalent amino acid residue thereof.
Peptides of formula I possess useful pharmaceutical properties. They are especially useful for treating disease states capable of being modulated by compounds which bind to parathyroid hormone receptors either with or without concommitant stimulation of cAMPase activity. See U.S. Ser. No. 60/046,472.
Representative preferred cyclic peptides suitable for preparation using the process of this invention include, but are not limited to
cyclo(Lys18-Asp22)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 1);
cyclo(Lys18-Asp22)[Ala1,2,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 2);
cyclo(Lys18-Asp22)[Ala1,3,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 3);
cyclo(Lys18-Asp22)[Ala1,4,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 4);
cyclo(Lys18-Asp22)[Ala1,5,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 5);
cyclo(Lys18-Asp22)[Ala1,6,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 6);
cyclo(Lys18-Asp22)[Ala1,7,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 7);
cyclo(Lys18-Asp22)[Ala1,8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 8);
cyclo(Lys18-Asp22)[Ala1,10,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 9);
cyclo(Lys18-Asp22)[Ala1,11,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 10);
cyclo(Lys18-Asp22)[Ala1,12,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 11);
cyclo(Lys18-Asp22)[Ala1,13,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 12);
cyclo(Lys18-Asp22)[Ala1,14,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 13);
cyclo(Lys18-Asp22)[Ala1,15,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 14);
cyclo(Lys18-Asp22)[Ala1,16,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 15);
bicyclo(Lys13-Asp17,Lys18-Asp22)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 16);
bicyclo(Lys18-Asp22,Lys26-Asp30)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 17);
cyclo(Lys18-Asp22)[Ala1,Nle8,Lys18Asp22,Leu27]hPTH(1-34)NH2 (SEQ ID NO: 18);
bicyclo(Lys13-Asp17,Lys18-Asp22)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1-34)NH2 (SEQ ID NO: 19);
bicyclo(Lys18-Asp22Lys26-Asp30)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-34)NH2(SEQ ID NO: 46);
cyclo(Lys18-Asp22)[Lys18,Asp22]hPTHrP(1-34)NH2 (SEQ ID NO: 20);
cyclo(Lys18-Asp22)[Lys18,26,30,Asp22,Leu23,28,31,Glu25,29]hPTHrP(1-34)NH2 (SEQ ID NO: 21);
cyclo(Lys18-Asp22)[Nle8,Lys18,Asp22,Leu27]hPTH(7-34)NH2 (SEQ ID NO: 22);
cyclo(Lys18-Asp22)[Lys18,Asp22]hPTHrP(7-34)NH2 (SEQ ID NO: 23);
bicyclo(Lys13,Asp17,Lys18,Asp22)[Nle8,Lys18,Asp22,Leu27]hPTH(7-34)NH2, and SEQ ID NO: 24); and
bicyclo(Lys18-Asp22,Lys26,Asp30)[Nle8,Lys18,Asp22,Leu27]hPTH(7-34)NH2 (SEQ ID NO: 25).
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein the amino acid residues are xcex1-amino acid residues having L stereochemistry at the xcex1-carbon.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein X is selected from the group consisting of (a) R1axe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94 wherein R1a is H or Pro, A1 is Ser or Ala, A2 is Val, A3 is Ser. A4 Glu, A5 is Ile or His, A6 is Gln, (b) R1bxe2x80x94, A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94 wherein A2, A3, A4, A5 and A6 are defined above and R1b is H, (c) R1bxe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94 wherein R1b, A3, A4, A5 and A6 are defined above, (d) R1bxe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94 wherein R1b, A4, A5 and A6 are defined above, (e) R1bxe2x80x94A5xe2x80x94A6xe2x80x94 wherein R1b, A5 and A6 are defined above, (f) R1bA6xe2x80x94 wherein R1b and A6 are defined above and (g) R1bxe2x80x94 wherein R1b is defined above; A7 is Leu; A8 is Leu or Nle; A9 is His; A10 is Asp or Asn; A11 is Leu; A12 is Gly; A13, A17, A18, A22 and A30 are independently selected from Ser, Thr, Lys, Cys, homo-Cys Orn, Asp, Glu, xe2x80x94NHCH(CH2NH2)COxe2x80x94, xe2x80x94NHCH[(CH2)2NH2]COxe2x80x94, xe2x80x94NHCH[(CH2)3CO2H]COxe2x80x94 and xe2x80x94NHCH[(CH2)4CO2H]COxe2x80x94; A14 is His or Ser; A15 is Ser or Leu; A16 is Asn or Gln; A19 is Arg or Glu; A20 is Arg; A21 is Arg or Val; A23 is Phe or Trp; A24 is Leu; A25 is Arg or His; A26 is His or is independently selected from the group consisting of Ser, Thr, Lys, Cys, homo-Cys, Orn, Asp, Glu, xe2x80x94NHCH(CH2NH2)COxe2x80x94, xe2x80x94NHCH[(CH2)2NH2]COxe2x80x94, xe2x80x94NHCH[(CH2)3CO2H]COxe2x80x94 and xe2x80x94NHCH[(CH2)4CO2H]COxe2x80x94; A27 is Leu or Lys; A28 is Ile or Leu; A29 is Ala or Gln; A31 is Ile or Val; and Y is selected from the group consisting of (a) xe2x80x94R3 wherein xe2x80x94R3 is xe2x80x94OH or xe2x80x94NR4R5 wherein R4 and R5 are independently selected from hydrogen and alkyl of one to four carbon atoms, (b) xe2x80x94A32xe2x80x94R3 wherein R3 is defined above and A32 is His, (c) xe2x80x94A32xe2x80x94A33xe2x80x94R3 wherein R3 and A32 are defined above and A33 is Asn or Thr and (d) xe2x80x94A32xe2x80x94A33xe2x80x94A34 wherein A34 is Ala or Phe.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein
(i) the side chains of A18 and A22 are linked through an amide bond,
(ii) the side chains of A13 and A17 are linked through an amide bond, and the side chains of A18 and A22 are linked through an amide bond,
(iii) the side chains of A18 and A22 are linked through an amide bond, and the side chains of A26 and A30 are linked through an amide bond or
(iv) the side chains of A13 and A17 are linked through an amide bond, the side chains of A18 and A22 are linked through an amide bond and the side chains of A26 and A30 are linked through an amide bond.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein A13 is selected from Lys and Ala, A17 is selected from Ser and Asp, A18 is Lys, A22 is Asp, A26 is Lys and A30 is Asp.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein X is R1axe2x80x94A1xe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein A1 is Ala, A8 is Nle and A27 is Leu.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein R1a is H and Y is NH2.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein R1a is H and Y is xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94NH2.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein X is selected from the group consisting of (a) R1bxe2x80x94A2xe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (b) R1bxe2x80x94A3xe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (c) R1bxe2x80x94A4xe2x80x94A5xe2x80x94A6xe2x80x94, (e) R1bxe2x80x94A6xe2x80x94 and (f) R1bxe2x80x94.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein A8 is Nle and A27 is Leu.
Another preferred cyclic peptide suitable for preparation using the process of this invention has formula I wherein X is H and Y is xe2x80x94A32xe2x80x94A33xe2x80x94A34xe2x80x94NH2.
More preferred cyclic peptides suitable for preparation using the process of this invention are selected from cyclo(Lys18-Asp22)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 1);
cyclo(Lys18-Asp22)[Ala1,2,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 2);
cyclo(Lys18-Asp22)[Ala1,3,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 3);
cyclo(Lys18-Asp22)[Ala1,4,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 4);
cyclo(Lys18-Asp22)[Ala1,5,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 5);
cyclo(Lys18-Asp22)[Ala1,6,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 6);
cyclo(Lys18-Asp22)[Ala1,7,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 7);
cyclo(Lys18-Asp22)[Ala1,8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 8);
cyclo(Lys18-Asp22)[Ala1,10,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 9);
cyclo(Lys18-Asp22)[Ala1,11,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 10);
cyclo(Lys18-Asp22)[Ala1,12,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 11);
cyclo(Lys18-Asp22)[Ala1,13,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 12);
cyclo(Lys18-Asp22)[Ala1,14,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 13);
cyclo(Lys18-Asp22)[Ala1,15,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 14);
cyclo(Lys18-Asp22)[Ala1,16,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 15);
bicyclo(Lys13-Asp17,Lys18-Asp22)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 16); and
bicyclo(Lys18-Asp22,Lys26,Asp30)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 17);
Still more preferred cyclic peptides suitable for preparation using the process of this invention are selected from
cyclo(Lys18-Asp22)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 1);
bicyclo(Lys13-Asp17,Lys18-Asp22)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 16); and
bicyclo(Lys18-Asp22,Lys26-Asp30)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 17);
A still yet more preferred cyclic peptide suitable for preparation using the process of this invention is
cyclo(Lys18-Asp22)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1-31)NH2 (SEQ ID NO: 1);
The foregoing may be better understood by reference to the following example, which is presented for illustration and is not intended to limit the scope of this invention.